- 學位論文
腎絲球足細胞在急性與慢性足細胞損傷中的變化
The response of glomerular podocytes to acute and chronic podocyte injury
摘要
足細胞在腎絲球過濾屏障中扮演著不可或缺的角色，當足細胞病變或數目減少時會導致蛋白尿發生，長期下來會造成腎絲球硬化，喪失功能，產生末期腎病。隨著全球糖尿病患者的盛行率愈來愈高，糖尿病腎病(diabetic kidney disease；DKD)也越發普遍，目前臨床上常利用蛋白尿作為診斷與評估糖尿病腎病進展的指標，當尿蛋白增加時往往代表足細胞的損傷或功能已經出現異常；然而在蛋白尿升高之前，腎絲球早已承受超過率(hyperfiltration)與微血管內壓力增加多時。因此，如若能在蛋白尿發生之前，更早期地了解足細胞的基因、形態與功能變化， 對於一些急性或慢性腎病的早期發現、治療與預防策略是十分重要的。 根據過去的研究發現，在腎絲球損傷時足細胞會與周遭的細胞進行信號溝通相互影響，例如:腎絲球內皮細胞受損時可能會導致足細胞受傷脫落，而足細胞的功能異常與喪失也會造成內皮細胞死亡。此外，受損但未脫落的足細胞可能因為基因變異而釋放出不正常的訊號進一步導致足突消失(foot effacement)、脫落、肥大、凋亡等現象，這些情況都可能是急性腎損傷轉為慢性腎病亦或者是糖尿病演變成糖尿病腎病的關鍵原因，因此我們想要知道在急性腎損傷後以及早期糖尿病尚未出現蛋白尿時的足細胞基因表現變化並觀察這兩個時期的腎絲球與足細胞的形態變化。 首先我們利用Podocin-cre;itdTomato;iDTR的基因改造小鼠進行實驗。此小鼠表現Podocin基因的足細胞因會表現Cre 重組酶而活化紅色螢光蛋白(tdTomato)與人類白喉毒素受體(diphtheria toxin receptor；DTR)，可藉此觀察腎絲球足細胞在損傷下的組織學變化，並有利於足細胞的分選收集與進一步分析。在急性腎絲球足細胞損傷的實驗中，我們給予特定劑量的白喉毒素注射後，小鼠約25%的足細胞會從基底膜上脫落，出現嚴重的蛋白尿(尿液中的白蛋白/肌酐酸比值會大幅上升)，但經過一段時間之後蛋白尿的嚴重程度會減輕，推測腎絲球中尚未脫落之足細胞或許發生了代償變化以致出現某些機制來改善腎絲球過濾屏障的功能以降低蛋白尿，之後將會收集這些受損的足細胞去做微陣列分析來判斷基因發生了哪些改變，以利後續實驗來探索改善腎絲球足細胞的功能，減輕或延緩腎絲球硬化的進展。 我們接著進行腎絲球足細胞慢性損傷的實驗。一開始先利用野生種(wild type) C57BL/6小鼠，給予鏈脲佐菌素(Streptozotocin；STZ) 誘導小鼠血糖上升產生糖尿病。實驗結果顯示在STZ誘導5週之後，腎絲球過濾率會升高，腎絲球體積也會增加；然而，到STZ誘導10週時並未觀察到腎絲球出現硬化，於足細胞上也沒有發現脫落情形，在這段期間尿液中的鹽分濃度與蛋白尿程度也未觀察到變化。我們接著會利用Podocin-cre;itdTomato小鼠，在給予STZ之後第五週時收集這些受腎絲球超過濾影響的足細胞去做微陣列分析來判斷基因發生了哪些改變，以利後續實驗來探索改善腎絲球足細胞的功能，減輕或延緩腎絲球硬化的進展。 總結來說，急性足細胞凋亡或慢性腎絲球壓力增加都會對於存活的足細胞造成壓力並可能產生病變，最終會使得腎絲球過濾功能改變，並導致腎絲球後續硬化的進展。我們已建立急性與慢性腎絲球足細胞凋亡與壓力增加的動物模式，進一步的研究將可釐清足細胞在急性或慢性的壓力下，其代償或病生理的改變，是否可能成為更早期的治療標的，以防止腎絲球硬化的進行。
並列摘要
Podocytes play an indispensable role in the glomerular filtration barrier. Podocytopathy or loss would lead to proteinuria, development of glomerulosclerosis, loss of glomerular filtration, and end stage renal disease (ESRD) in the long term. With the increasing prevalence of diabetic patients around the world, more and more diabetic kidney disease (DKD) arises. The occurrence of proteinuria always represents injury or dysfunction of glomerular filtration barrier, which has been applied to diagnose the progression of DKD in the past. However, glomerulus has already born hyperfiltration and rising capillary pressure for a long time before proteinuria occurred. Therefore, if we could unveil the changes of transcriptome, histology and function in podocytes early enough before proteinuria occurs, we would have a great chance to diagnose and treat glomerulopathy as early as possible. Evidence has shown that podocytes will cross talk with surrounding cells including endothelial cells and mesangial cells while glomerulus is damaged. For instance, the injured glomerular endothelial cells may contribute to podocyte loss, and vice versa podocyte detachment could cause endothelial cell death. In addition, the podocytes may respond to injury by foot process effacement, hypertrophy, apoptosis or detachment from basement membrane, leading to transition of acute kidney injury into chronic kidney disease as well as the development of DKD in diabetes patients. Thus in this study, we would like to establish the models of acute podocyte ablation and glomerular hyperfiltration by specific genetic ablation and induction of diabetes mellitus respectively to unveil the response of the podocytes to acute injury or chronic hyperfiltration through transcriptome and histological analyses of podocytes. We first used Podocin-cre;itdTomato;iDTR genetically modified mice to carry out our experiments. The podocytes of the mice expressed Podocin gene could activate red fluorescence protein (tdTomato) and diphtheria toxin receptor (DTR) through Cre recombinase expressing. It is useful to observe the histological changes of glomerular podocytes after injury and to sort the cells by tdTomato expression. First, we carried out the experiments of acute podocyte injury by diphtheria toxin (DT) injection. After specific DT dose injected 14 days, 25% of glomerular podocytes would be ablated. Besides, severe proteinuria assessed by urine albumin/creatinine ratio would happen. Nevertheless, the proteinuria had improved since day 14 after DT injection, suggesting that surviving podocytes might have some compensatory mechanisms to improve glomerular filtration barrier and proteinuria. Next, we will sort these surviving podocytes to analyze the transcriptome by microarray. Secondly we carried out the experiments of chronic glomerular podocyte injury by inducing glomerular hyperfiltration. We first injected Streptozotocin (STZ) to wild type C57BL/6 mice in order to induce high blood sugar and diabetes. The results showed that the glomerular filtration rate assessed by inulin clearance raised and the glomerular volume increased 5 weeks after STZ injection. Nonetheless, the glomerulosclerosis and detachment of podocytes did not appear in Periodic acid-Schiff and immunofluorescence staining after STZ injected 10 weeks. Additionally, the urinary salt excretion and proteinuria did not change during the period. Next, we will use Podocin-cre;itdTomato mice for diabetes induction and sort the tdTomato+ podocytes at 5 weeks after STZ injection when hyperfiltration has happened and impacted on the podocytes. The sorted podocytes will be analyzed for transcriptome by microarray. Through these transcriptome analyses, we expect to unveil the mechanisms of surviving podocytes compensating the podocyte loss and the response of podocytes to hyperfiltration. In conclusion, acute podocytes apoptosis or chronic increase of glomerular pressure impact on the surviving podocytes. In the end, the glomerular filtration function would change and lead to subsequent progression of glomerulosclerosis. We have established the models of acute and chronic podocyte injuries, and we will use these models to unveil the mechanisms of podocytes compensating the cell loss and adapting to increased glomerular pressure in this study. We expect the study might provide early therapeutic targets to prevent glomerulosclerosis.